Treatment of cancer with elevated dosages of soluble fgfr1 fusion proteins

ABSTRACT

The present invention provides methods of treating a patient having a cancer comprising administering to the patient a soluble Fibroblast Growth Factor Receptor 1 (FGFR1) fusion protein such as an extracellular domain of an FGFR1 polypeptide linked to an Fc polypeptide or another fusion partner. The fusion protein may be administered at a dose of at least about 2 mg/kg body weight. In some embodiments, the patient has a fibroblast growth factor-2 (FGF-2) plasma concentration of at least 6 pg/ml. In some embodiments, the cancer is characterized by a Fibroblast Growth Factor Receptor 2 (FGFR 2 ) having a ligand-dependent activating mutation.

RELATED APPLICATIONS

This application is a non-provisional application filed under 37 CFR1.53(b)(1), claiming priority under 35 USC 119(e) to provisionalapplication No. 61/413,940 filed Nov. 15, 2010, the content of which isincorporated herein by reference.

BACKGROUND AND SUMMARY OF THE INVENTION

The Fibroblast Growth Factor (FGF)-Fibroblast Growth Factor Receptor(FGFR) signaling pathway is widely implicated in the development andmaintenance of many different cancers. This signaling pathway comprises4 different FGF receptors (FGFR1, FGFR2, FGFR3, and FGFR4), some ofwhich are also alternatively spliced, and 22 different FGF ligands. EachFGF receptor and splice form has different patterns of expression anddifferent specificities for the various FGF ligands.

FGFR1 is the best characterized of the four FGFRs. FGFR1 and its ligandshave causal connections to cancer in animal models and strongcorrelative connections to human disease. Specific induction of FGFR1signaling in mouse prostate results in prostate hyperplasia andcarcinoma (Freeman et al., Cancer Res. 2003; 63:8256-63), demonstratingthat abnormal hyperactivation of FGFR1 is sufficient to initiatetumorigenesis. Inhibition of FGFR1 activity inhibits tumor growth inxenograft models from multiple tissue types (Ogawa et al., Cancer GeneTher. 2002; 9:633-40). In human disease, chromosomal amplification ofthe FGFR1 gene in a subset of breast cancer patients is associated withpoor outcome (Gelsi-Boyer et al., Mol. Cancer. Res. 2005; 3:655-67) andoverexpression or high systemic levels of FGF ligands correlate withtumorigenesis and poor patient outcome (Nguyen et al., J. Natl. CancerInst. 1994; 86:356-61).

FGFR1 has multiple mechanisms in the promotion of tumor cell growth andsurvival. FGFR1 signaling increases the mitotic rate of tumor cells,promotes tumor angiogenesis, and helps maintain the tumorigenicity oftumor stem cells (TSCs). Many tumor cell lines are responsive to anddependent on FGFR1 signaling for growth in vitro, and tumor cell linesbecome resistant to cytotoxic agents when stimulated with FGF-2 (Song etal., PNAS 2000; 97:8658-63). Disruption of the FGF-FGFR1 pathway leadsto reduction of tumor cell growth in vitro and in xenografts (Ogawa etal., Cancer Gene Ther. 2002; 9:633-40). Specific inhibition of FGFRsignaling may therefore cause reduction in the growth rate of humantumors.

Some FGFs have potent angiogenic activities and play important roles intumor vasculogenesis (Compagni et al., Cancer Res. 2000; 60:7163-69). Inmurine models of pancreatic cancer, FGFs mediate escape fromanti-vascular endothelial growth factor receptor (VEGFR) therapy. Asimilar phenomenon is seen in glioblastoma multiforme patients treatedwith anti-VEGFR therapy, in whom tumor progression correlates withgreatly increased levels of FGF-2 (Batchelor et al., Cancer Cell 2006;11:83-95). Inhibition of FGFR1 might reduce tumor angiogenesis,particularly in tumors previously treated with anti-VEGF therapies.

There is increasing evidence that tumors contain a small population ofmalignant cells (TSCs) that are phenotypically similar to stem cellsthat may be responsible for tumor initiation, survival, proliferation,and recurrence. These cells are dependent on the presence of FGFs tomaintain their TSC phenotypes (Dvorak et al., FEBS Letters 2006;580:2869-74). When FGFs are withdrawn from in vitro cultures of TSCs,they stop proliferating and appear to differentiate. Thus, inhibition ofFGFR1 signaling may lead to reduced metastases and recurrence of tumors.

Soluble FGFR1 fusion proteins are able to bind to FGF ligands of theFGFR1 receptor, “trapping” the ligands and prevent them from activatingFGFR1 receptors as well as other receptors for which the ligands haveaffinity. See, e.g., U.S. Pat. No. 7,678,890. Without being bound to aparticular theory, it is believed that soluble FGFR1 fusion proteins caninhibit tumorigenic activity through multiple mechanisms of action,including but not limited to, direct anti-tumor activity in cancersdependent on the FGF-FGFR pathway, inhibition of tumor angiogenesis,and/or inhibition of cancer stem cell maintenance.

The data provided here show for the first time that a soluble FGFR1/Fcfusion protein, FP-1039, can be administered safely to human patients atconcentrations of about 2 mg/kg body weight or higher (i.e., up to atleast about 16 mg/kg) and that such concentrations are well-tolerated.As shown in detail in the Examples, in some embodiments, treatment ofhumans with FP-1039 yields pharmacokinetic and pharmacodynamic profilesthat indicate weekly or less frequent administration of doses above 2mg/kg, 4 mg/kg, 8 mg/kg or 10 mg/kg is sufficient for sustainedsequestration of target FGF ligands such as FGF-2.

In one aspect, the present invention provides methods of treating ahuman having a cancer. In some embodiments, the method comprisesadministering to the human a therapeutically effective amount of asoluble Fibroblast Growth Factor Receptor 1 (FGFR1) fusion protein,wherein the fusion protein comprises an extracellular domain of an FGFR1polypeptide linked to a fusion partner. In some embodiments, FGFR1fusion protein is administered at a dose of about 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 mg/kg body weight, or within arange from one to another of the above dose values (the above dosesbeing calculated using an extinction coefficient of 1.42 mL/mg*cm). Insome embodiments, the FGFR1 fusion protein is administered at a dose ofabout 10 mg/kg body weight as calculated using an extinction coefficientof 1.11 mL/mg*cm. In other embodiments, the FGFR1 fusion protein isadministered at a dose of about 20 mg/kg body weight as calculated usingan extinction coefficient of 1.11 mL/mg*cm, or at a range of about 10 toabout 20 mg/kg body weight as calculated using an extinction coefficientof 1.11 mL/mg*cm. In some embodiments, the human has a fibroblast growthfactor-2 (FGF-2) plasma concentration of at least 6 pg/ml. In someembodiments, the cancer is characterized by a ligand-dependentactivating mutation in FGFR2. In some embodiments, the ligand-dependentactivating mutation in FGFR2 is S252W or P253R. In some embodiments, thesoluble FGFR1 fusion protein is administered in combination with achemotherapeutic agent or a VEGF antagonist.

In some embodiments, the FGFR1 polypeptide is human FGFR1 isoform IIIc.In some embodiments, the fusion partner is an Fc polypeptide, which isthe Fc region of human immunoglobulin G1 (IgG1). In some embodiments,the FGFR1 extracellular domain has the amino acid sequence of SEQ IDNO:5. In some embodiments, the soluble FGFR1 fusion protein has theamino acid sequence of SEQ ID NO:8.

In some embodiments, the soluble FGFR1 fusion protein is administered ata dose of about 2 mg/kg body weight to about 30 mg/kg body weight. Insome embodiments, the soluble FGFR1 fusion protein is administered at adose of about 8 mg/kg body weight to about 16 mg/kg body weight (orabout 10 mg/kg body weight to about 20 mg/kg body weight when calculatedusing an extinction coefficient of 1.11 mL/mg*cm). In some embodiments,the soluble FGFR1 fusion protein is administered at a dose of about 8mg/kg body weight, while in some embodiments, the soluble FGFR1 fusionprotein is administered at a dose of about 16 mg/kg body weight (or atabout 10 mg/kg body weight or about 20 mg/kg body weight, respectively,when calculated using an extinction coefficient of 1.11 mL/mg*cm).

In some embodiments, the method comprises administering FP-1039 to ahuman patient having cancer, wherein the human has a fibroblast growthfactor-2 (FGF-2) plasma concentration of at least 6 pg/ml and whereinFP-1039 is administered at a dose of about 2 mg/kg to about 30 mg/kg. Insome embodiments, the soluble FGFR1 fusion protein is administered at adose of about 8 mg/kg body weight. In some embodiments, the FP-1039 isadministered at about 16 mg/kg.

In some embodiments, the soluble FGFR1 fusion protein is administeredtwice a week, weekly, every other week, at a frequency between weeklyand every other week, every three weeks, every four weeks, or everymonth.

In some embodiments, the soluble FGFR1 fusion protein is administeredintravenously or subcutaneously.

In some embodiments, the cancer is prostate cancer, breast cancer,colorectal cancer, lung cancer, endometrial cancer, head and neckcancer, laryngeal cancer, liver cancer, renal cancer, glioblastoma, orpancreatic cancer.

In some embodiments, the human has an FGF-2 plasma concentration of atleast 10 pg/ml prior to the administration of the soluble FGFR1 fusionprotein. In some embodiments, the soluble FGFR1 fusion protein isadministered at a dose such that at seven days after administration, thehuman has an FGF-2 plasma concentration of less than 4 pg/ml.

In some embodiments, the soluble FGFR1 fusion protein is administered incombination with a chemotherapeutic agent. In some embodiments, thechemotherapeutic agent is sorafenib. In some embodiments, the solubleFGFR1 fusion protein is administered in combination with a VEGFantagonist. In some embodiments, the VEGF antagonist is a VEGF antibody,such as bevacizumab, or the VEGF antagonist is a VEGF trap, such asaflibercept. In some embodiments, the soluble FGFR1 fusion protein isadministered in combination with an anti-angiogenic agent.

The present invention also provides for methods of treating a humanhaving a cancer, wherein the cancer is characterized by an FibroblastGrowth Factor Receptor 2 (FGFR2) having a ligand-dependent activatingmutation, the method comprising: administering to the human a solubleFibroblast Growth Factor Receptor 1 (FGFR1) fusion protein at a dose ofabout 2 mg/kg body weight to about 30 mg/kg body weight, wherein thefusion protein comprises an extracellular domain of an FGFR1 polypeptidelinked to a Fc polypeptide. In some embodiments, the soluble FGFR1fusion protein is administered at a dose of about 8 mg/kg body weight,while in some embodiments, the FP-1039 is administered at about 16 mg/kgbody weight (or about 10 mg/kg body weight or about 20 mg/kg bodyweight, respectively when calculated using an extinction coefficient of1.11 mL/mg*cm).

In some embodiments wherein the cancer is characterized by an FGFR2having a ligand-dependent activating mutation, the FGFR1 polypeptide ishuman FGFR1 isoform IIIc. In some embodiments wherein the cancer ischaracterized by an FGFR2 having a ligand-dependent activating mutation,the Fc polypeptide is an Fc region of human immunoglobulin G1 (IgG1).

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the FGFR1 extracellular domaincomprises the amino acid sequence of SEQ ID NO:5. In some embodiments,the soluble FGFR1 fusion protein comprises the amino acid sequence ofSEQ ID NO:8.

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the soluble FGFR1 fusion proteinis administered at a dose of about 2 mg/kg body weight to about 30 mg/kgbody weight. In some embodiments for treating cancer characterized by anFGFR2 having a ligand-dependent activating mutation, the soluble FGFR1fusion protein is administered at a dose of about 8 mg/kg body weight toabout 16 mg/kg body weight (or about 10 mg/kg body weight to about 20mg/kg body weight when calculated using an extinction coefficient of1.11 mL/mg*cm). In some embodiments for treating cancer characterized byan FGFR2 having a ligand-dependent activating mutation, the solubleFGFR1 fusion protein is administered at a dose of about 8 mg/kg bodyweight, while in some embodiments for treating cancer characterized byan FGFR2 having a ligand-dependent activating mutation, the solubleFGFR1 fusion protein is administered at a dose of about 16 mg/kg bodyweight (or about 10 mg/kg body weight or about 20 mg/kg body weight,respectively when calculated using an extinction coefficient of 1.11mL/mg*cm). In some embodiments for treating cancer characterized by anFGFR2 having a ligand-dependent activating mutation, the FGFR1 fusionprotein is administered at a dose of about 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 25, 26, 28 or 30 mg/kg body weight, or within a rangefrom one to another of the above dose values. In some embodiments,dosages may be administered weekly, every other week, at a frequencybetween weekly and every other week, every three weeks, every fourweeks, or every month.

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the method comprisesadministering FP-1039 to a human patient having cancer, wherein thecancer is characterized by an FGFR2 having a ligand-dependent activatingmutation, and wherein FP-1039 is administered at a dose of about 2 mg/kgto about 30 mg/kg. In some embodiments, the FP-1039 is administered atabout 8 mg/kg, while in some embodiments, the FP-1039 is administered atabout 16 mg/kg (or about 10 mg/kg body weight to about 20 mg/kg bodyweight when calculated using an extinction coefficient of 1.11mL/mg*cm).

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the soluble FGFR1 fusion proteinis administered weekly or every other week or a frequency between weeklyand every other week.

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the soluble FGFR1 fusion proteinis administered intravenously or subcutaneously.

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the cancer is prostate cancer,breast cancer, colorectal cancer, lung cancer, endometrial cancer, headand neck cancer, laryngeal cancer, liver cancer, renal cancer,glioblastoma, or pancreatic cancer.

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the human has an FGF-2 plasmaconcentration of at least 10 pg/ml prior to the administration of thesoluble FGFR1 fusion protein. In some embodiments for treating cancercharacterized by an FGFR2 having a ligand-dependent activating mutation,the soluble FGFR1 fusion protein is administered at a dose such that atseven days after administration, the human has an FGF-2 plasmaconcentration of less than 4 pg/ml.

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the soluble FGFR1 fusion proteinis administered in combination with a chemotherapeutic agent. In someembodiments, the chemotherapeutic agent is sorafenib. In someembodiments for treating cancer characterized by an FGFR2 having aligand-dependent activating mutation, the soluble FGFR1 fusion proteinis administered in combination with a chemotherapeutic agent, VEGFantagonist or anti-angiogenic agent. In some embodiments, the VEGFantagonist is a VEGF antibody, such as bevacizumab, or the VEGFantagonist is a VEGF trap, such as aflibercept. In some embodiments, thesoluble FGFR1 fusion protein is administered in combination with ananti-angiogenic agent.

In some embodiments for treating cancer characterized by an FGFR2 havinga ligand-dependent activating mutation, the ligand-dependent activatingmutation in FGFR2 is S252W or P253R.

The present invention also provides a composition comprising a solubleFGFR1 fusion protein for use in the treatment of cancer, wherein thecomposition is administered at a dose of at least about 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 mg/kg body weight, orwithin a range from one to another of the above dose values. In someembodiments, dosages may be administered twice a week, weekly, everyother week, at a frequency between weekly and every other week, everythree weeks, every four weeks, or every month. In some embodiments, thehuman has a fibroblast growth factor-2 (FGF-2) plasma concentration ofat least 6 pg/ml. In some embodiments, the cancer is characterized by aligand-dependent activating mutation in FGFR2. In some embodiments, theligand-dependent activating mutation in FGFR2 is S252W or P253R. In someembodiments, the soluble FGFR1 fusion protein is administered incombination with a chemotherapeutic agent or a VEGF antagonist.

Any embodiment described herein or any combination thereof applies toany and all methods of the invention described herein.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

In this application, the use of “or” means “and/or” unless statedotherwise. In the context of a multiple dependent claim, the use of “or”refers back to more than one preceding independent or dependent claim inthe alternative only.

As used herein, all numbers are approximate, and may be varied toaccount for measurement error and the rounding of significant digits.The use of “about” before certain measured quantities includesvariations due to sample impurities, measurement error, human error, andstatistical variation, as well as the rounding of significant digits.

As used herein, a “fibroblast growth factor receptor 1” or “FGFR1”polypeptide refers to a polypeptide having the amino acid sequence ofany one of the known FGFR1 polypeptides, such as FGFR1-IIIb andFGFR1-IIIc, and any variant, precursor, or fragment thereof, includingthose described in U.S. Pat. Nos. 7,678,890; 6,656,728; 6,384,191;6,255,454; 6,344,546; 5,288,855; and 5,229,501. An FGFR1 polypeptidesequence is typically from, or derived from, a mammal including, but notlimited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster;cow; pig; sheep; horse; or any other mammal.

As used herein, “extracellular domain” or “ECD” refers to the portion ofa polypeptide that extends beyond the transmembrane domain of thepolypeptide into the extracellular space. In some embodiments, the ECDis an ECD of an FGFR1 polypeptide, such as FGFR1-IIIb and FGFR1-IIIc, ora variant thereof. The term “FGFR1 extracellular domain” (“FGFR1 ECD”)includes full-length FGFR1 ECDs, FGFR1 ECD fragments, and FGFR1 ECDvariants. As used herein, the term “FGFR1 ECD” refers to an FGFR1polypeptide that lacks the intracellular and transmembrane domains, withor without a signal peptide. In some embodiments, the FGFR1 ECDcomprises an amino acid sequence that is substantially identical to anECD having the amino acid sequence of SEQ ID NO:1. In some embodiments,the FGFR1 ECD has the amino acid sequence of any of SEQ ID NOs:1-6.

As used herein, a “soluble FGFR fusion protein” or an “FGFR1 fusionprotein” or “FGFR1 ECD fusion molecule” refers to a protein comprisingan FGFR1 ECD linked to one or more fusion partners, wherein the solubleFGFR fusion protein lacks a transmembrane domain (e.g., an FGFR1transmembrane domain) and is not bound to the cellular membrane.

As used herein, a “fusion partner” is a molecule that is linked to theFGFR1 ECD that imparts favorable pharmacokinetics and/orpharmacodynamics on the FGFR1 ECD protein. A fusion partner may comprisea polypeptide, such as a fragment of an immunoglobulin molecule oralbumin, or it may comprise a non-polypeptide moiety, for example,polyethylene glycol. In some embodiments, the fusion partner is an Fcdomain of an antibody.

The term “signal peptide” refers to a sequence of amino acid residueslocated at the N terminus of a polypeptide that facilitates secretion ofa polypeptide from a mammalian cell. A signal peptide may be cleavedupon export of the polypeptide from the mammalian cell, forming a matureprotein. Signal peptides may be natural or synthetic, and they may beheterologous or homologous to the protein to which they are attached.Exemplary signal peptides also include signal peptides from heterologousproteins. A “signal sequence” refers to a polynucleotide sequence thatencodes a signal peptide. In some embodiments, an FGFR1 ECD lacks asignal peptide. In some embodiments, an FGFR1 ECD includes at least onesignal peptide, which may be a native FGFR1 signal peptide or aheterologous signal peptide.

A “ligand-dependent activating mutation” of FGFR2 refers to a mutationthat increases the biological activity of FGFR2, for example, a mutationcausing FGFR2 to become activated more readily in comparison to thewildtype FGFR2 in response to certain stimuli, wherein the biologicaleffects of the mutation depend on the binding of FGFR2 to one or more ofits ligands. An example is a mutation that causes alterations in theligand binding properties of FGFR2.

As used herein, the terms “native FGFR1 ECD” and “wildtype FGFR1 ECD”are used interchangeably to refer to an FGFR1 ECD with a naturallyoccurring amino acid sequence. Native FGFR1 ECDs and wildtype FGFR1 ECDsalso include FGFR1 ECD splice variants or isoforms. As used herein, theterms FGFR1 ECD “splice variants” or “splice isoforms” are usedinterchangeably to refer to alternative splice forms of FGFR1 ECD, suchas FGFR1-IIIb and FGFR1-IIIc ECD.

As used herein, the term “FGFR1 ECD variants” refers to FGFR1 ECDscontaining amino acid additions, deletions, and/or substitutions incomparison to the native FGFR1 ECDs. FGFR1 ECD variants retain theability to bind FGF2. Such variants may be at least 90%, 92%, 95%, 97%,98%, or 99% identical to the parent FGFR1 ECD.

The term “full-length FGFR1 ECD”, as used herein, refers to an FGFR1 ECDthat extends to the last amino acid of the extracellular domain, and mayor may not include an N-terminal signal peptide. As used herein, theterm “FGFR1 ECD fragment” refers to an FGFR1 ECD having an amino acidsequence modified in that amino acid residues have been deleted from theamino-terminus and/or from the carboxy-terminus of the polypeptide,wherein the fragment retains the ability to bind FGF2. As used herein,the term “native FGFR1 ECD fragment” refers to an FGFR1 ECD fragment inwhich the retained portions of the FGFR1 ECD sequence are naturallyoccurring, wherein the fragment retains the ability to bind FGF2.

As used herein, the terms “FGFR1 ECD fragment variant” and “variant ofFGFR1 ECD fragment” are used interchangeably to refer to FGFR1 ECDscontaining, not only amino acid deletions from the amino- and/orcarboxy-terminus of native FGFR1 ECD, but also amino acid additions,deletions, and/or substitutions within the retained portion of the FGFR1ECD. FGFR1 ECD fragment variants also retain the ability to bind FGF2.

The terms “nucleic acid” or “polynucleotide” refer todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides which have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992);Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleicacid is used interchangeably with gene, cDNA, and mRNA encoded by agene.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer. The terms alsoinclude post-translational modifications of the polypeptide, including,for example, glycosylation, sialylation, acetylation, andphosphorylation. When a polypeptide “consists of” a particular aminoacid sequence, it may still contain post-translational modifications,such as glycosylation and sialylation.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., a γ-carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a number of nucleic acid sequences will encode anygiven protein. For instance, the codons GCA, GCC, GCG and GCU all encodethe amino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and

8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984))

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (e.g., a polypeptide of the invention), which doesnot comprise additions or deletions, for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same sequences. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over aspecified region, or, when not specified, over the entire sequence of areference sequence), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. The invention providespolypeptides that are substantially identical to the polypeptidesexemplified herein (e.g., the polypeptides exemplified in SEQ IDNOs:1-8). Optionally, the identity exists over a region that is at leastabout 15, 25 or 50 nucleotides in length, or more preferably over aregion that is 100 to 500 or 1000 or more nucleotides in length, or overthe full length of the reference sequence. With respect to amino acidsequences, identity or substantial identity can exist over a region thatis at least 5, 10, 15 or 20 amino acids in length, optionally at leastabout 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally atleast about 150, 200 or 250 amino acids in length, or over the fulllength of the reference sequence. With respect to shorter amino acidsequences, e.g., amino acid sequences of 20 or fewer amino acids,substantial identity exists when one or two amino acid residues areconservatively substituted, according to the conservative substitutionsdefined herein.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two polypeptides are substantially identical is thatthe first polypeptide is immunologically cross reactive with theantibodies raised against the second polypeptide. Thus, a polypeptide istypically substantially identical to a second polypeptide, for example,where the two peptides differ only by conservative substitutions.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. In some embodiments, cancer is a human cancer.Examples of cancer include but are not limited to carcinomas, sarcomas,adenocarcinomas, lymphomas, leukemias, solid and lymphoid cancers, etc.Examples of different types of cancer include, but are not limited to,pancreatic cancer, breast cancer, gastric cancer, bladder cancer, oralcancer, ovarian cancer, thyroid cancer, lung cancer (non-small cell lungcancer, small cell lung cancer, squamous cell lung cancer), (non-smallcell lung cancer, small cell lung cancer, squamous cell lung cancer),prostate cancer, uterine cancer, endometrial cancer, testicular cancer,neuroblastoma, squamous cell carcinoma of the head, neck, cervix andvagina, multiple myeloma, soft tissue and osteogenic sarcoma, colorectalcancer, liver cancer (i.e., hepatocarcinoma), renal cancer (i.e., renalcell carcinoma), mesothelioma, cervical cancer, anal cancer, bile ductcancer, gastrointestinal carcinoid tumors, gastrointestinal stromaltumors (GIST), esophageal cancer, laryngeal cancer, gall bladder cancer,small intestine cancer, cancer of the central nervous system, skincancer, choriocarcinoma; osteogenic sarcoma, rhabdomyosarcoma,fibrosarcoma, glioma, glioblastoma, melanoma, B-cell lymphoma,non-Hodgkin's lymphoma, Burkitt's lymphoma, Small Cell lymphoma, LargeCell lymphoma, monocytic leukemia, myelogenous leukemia, acutelymphocytic leukemia, and acute myelocytic leukemia. Cancers embraced inthe current application include both metastatic and non-metastaticcancers.

The terms “treating” or “treatment” refers to inhibiting a disease,i.e., arresting or reducing the development of the disease or itsclinical symptoms; or relieving a disease, for example, by causingregression, or restoring or repairing a lost, missing, or defectivefunction; or stimulating an inefficient process. In some embodiments,“treatment” refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.

The terms “subject” and “patient” are used interchangeably herein torefer to mammals, including, but not limited to, rodents, simians,humans, felines, canines, equines, bovines, porcines, ovines, caprines,mammalian laboratory animals, mammalian farm animals, mammalian sportanimals, and mammalian pets. In some embodiments, the subject is ahuman.

A “pharmaceutically acceptable carrier” refers to a solid, semisolid, orliquid filler, diluent, encapsulating material, formulation auxiliary,or carrier conventional in the art for use with a therapeutic agent foradministration to a subject. A pharmaceutically acceptable carrier isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation.Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition.

“FP-1039” refers to a protein having the amino acid sequence set forthin SEQ ID NO:8. FP-1039 can be produced, for example, as is describedgenerally for FGFR fusion molecules in U.S. Pat. No. 7,678,890, theentire disclosure of which is expressly incorporated herein byreference.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent.” Examples of therapeutic agents (anti-cancer agents)include, but are not limited to, e.g., chemotherapeutic agents, growthinhibitory agents, cytotoxic agents, agents used in radiation therapy,anti-angiogenic agents, apoptotic agents, anti-tubulin agents, andother-agents to treat cancer, such as anti-VEGF antibodies (e.g.,bevacizumab, AVASTIN®), anti-HER-2 antibodies (e.g., trastuzumab,HERCEPTIN®), anti-CD20 antibodies (e.g., rituximab, RITUXAN®), anepidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosinekinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVA®),platelet derived growth factor inhibitors (e.g., GLEEVEC® (ImatinibMesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines,antagonists (e.g., neutralizing antibodies) that bind to one or more ofthe following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMAor VEGF receptor(s), TRAIL/Apo2, and other bioactive and organicchemical agents, etc. Combinations thereof are also included in theinvention.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), pemetrexed(ALIMTA®); tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone,and 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®),albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE),and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,ELOXATIN®), and carboplatin; vincas, which prevent tubulinpolymerization from forming microtubules, including vinblastine(VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), andvinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone;leucovorin; novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid, including bexarotene(TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS®or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronicacid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g.,ABARELIX®); BAY439006 (sorafenib, NEXAVAR®; Bayer); SU-11248 (sunitinib,SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib oretoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®);CCl-779; tipifarnib (R11577); orafenib, ABT510; Bc1-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (seedefinition below); tyrosine kinase inhibitors (see definition below);serine-threonine kinase inhibitors such as rapamycin (sirolimus,RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636,SARASAR); and pharmaceutically acceptable salts, acids or derivatives ofany of the above; as well as combinations of two or more of the abovesuch as CHOP, an abbreviation for a combined therapy ofcyclophosphamide, doxorubicin, vincristine, and prednisolone; andFOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens with mixed agonist/antagonist profile, including,tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®),idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, andselective estrogen receptor modulators (SERMs) such as SERM3; pureanti-estrogens without agonist properties, such as fulvestrant(FASLODEX®), and EM800 (such agents may block estrogen receptor (ER)dimerization, inhibit DNA binding, increase ER turnover, and/or suppressER levels); aromatase inhibitors, including steroidal aromataseinhibitors such as formestane and exemestane (AROMASIN®), andnonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®),letrozole (FEMARA®) and aminoglutethimide, and other aromataseinhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®),fadrozole, and 4(5)-imidazoles; lutenizing hormone-releasing hormoneagonists, including leuprolide (LUPRON® and ELIGARD®), goserelin,buserelin, and tripterelin; sex steroids, including progestins such asmegestrol acetate and medroxyprogesterone acetate, estrogens such asdiethylstilbestrol and premarin, and androgens/retinoids such asfluoxymesterone, all transretinoic acid and fenretinide; onapristone;anti-progesterones; estrogen receptor down-regulators (ERDs);anti-androgens such as flutamide, nilutamide and bicalutamide; andpharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promote angiogenesis, endothelialcell growth, stability of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family (VEGF-B, VEGF-C and VEGF-D), P1GF, PDGFfamily, fibroblast growth factor family (FGFs), TIE ligands(Angiopoietins), ephrins, delta-like ligand 4 (DLL4), del-1, fibroblastgrowth factors: acidic (aFGF) and basic (bFGF), follistatin, granulocytecolony-stimulating factor (G-CSF), hepatocyte growth factor(HGF)/scatter factor (SF), interleukin-8 (IL-8), leptin, midkine,neuropilins, placental growth factor (P1GF), platelet-derivedendothelial cell growth factor (PD-ECGF), platelet-derived growthfactor, especially PDGF-BB or PDGFR-beta, Pleiotrophin (PTN),progranulin, proliferin, transforming growth factor-alpha (TGF-alpha),transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha(TNF-alpha), etc. It would also include factors that accelerate woundhealing, such as growth hormone, insulin-like growth factor-I (IGF-I),VIGF, epidermal growth factor (EGF), CTGF and members of its family, andTGF-alpha and TGF-beta. See, e.g., Klagsbrun and D'Amore (1991) Annu.Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179;Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al.(2003) Oncogene 22:6549-6556 (e.g., Table 1 listing known angiogenicfactors); and, Sato (2003) Int. J. Clin. Oncol. 8:200-206.

An “anti-angiogenic agent” or “angiogenesis inhibitor” refers to a smallmolecular weight substance, a polynucleotide (including, e.g., aninhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, arecombinant protein, an antibody, or conjugates or fusion proteinsthereof, that inhibits angiogenesis, vasculogenesis, or undesirablevascular permeability, either directly or indirectly. It should beunderstood that the anti-angiogenic agent includes those agents thatbind and block the angiogenic activity of the angiogenic factor or itsreceptor. For example, an anti-angiogenic agent is an antibody or otherantagonist to an angiogenic agent as defined above, e.g., fusionproteins that binds to VEGF-A such as ZALTRAP™ (Aflibercept), antibodiesto VEGF-A such as AVASTIN® (bevacizumab) or to the VEGF-A receptor(e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such asGLEEVEC® (Imatinib Mesylate), small molecules that block VEGF receptorsignaling (e.g., PTK787/ZK2284, SU6668, SUTENT®/SU11248 (sunitinibmalate), AMG706, or those described in, e.g., international patentapplication WO 2004/113304). Anti-angiogenic agents also include nativeangiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g.,Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit andDetmar (2003) Oncogene 22:3172-3179 (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo (1999)Nature Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene22:6549-6556 (e.g., Table 2 listing known anti-angiogenic factors); and,Sato (2003) Int. J. Clin. dOncol. 8:200-206 (e.g., Table 1 listinganti-angiogenic agents used in clinical trials).

The term “VEGF” or “VEGF-A” as used herein refers to the 165-amino acidhuman vascular endothelial cell growth factor and related 121-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by Leung et al. (1989) Science 246:1306, and Houck et al.(1991) Mol. Endocrin, 5:1806, together with the naturally occurringallelic and processed forms thereof. The term “VEGF” also refers toVEGFs from non-human species such as mouse, rat or primate. Sometimesthe VEGF from a specific species are indicated by terms such as hVEGFfor human VEGF, mVEGF for murine VEGF, and etc. The term “VEGF” is alsoused to refer to truncated forms of the polypeptide comprising aminoacids 8 to 109 or 1 to 109 of the 165-amino acid human vascularendothelial cell growth factor. Reference to any such forms of VEGF maybe identified in the present application, e.g., by “VEGF (8-109),” “VEGF(1-109),” “VEGF-A₁₀₉” or “VEGF165.” The amino acid positions for a“truncated” native VEGF are numbered as indicated in the native VEGFsequence. For example, amino acid position 17 (methionine) in truncatednative VEGF is also position 17 (methionine) in native VEGF. Thetruncated native VEGF has binding affinity for the KDR and Flt-1receptors comparable to native VEGF.

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including, but not limited to, its binding to one or moreVEGF receptors. VEGF antagonists include, without limitation, anti-VEGFantibodies and antigen-binding fragments thereof, receptor molecules andderivatives which bind specifically to VEGF thereby sequestering itsbinding to one or more receptors, anti-VEGF receptor antibodies, VEGFreceptor antagonists such as small molecule inhibitors of the VEGFRtyrosine kinases, and immunoadhesins that bind to VEGF such as VEGFtraps (e.g., aflibercept). The term “VEGF antagonist,” as used herein,specifically includes molecules, including antibodies, antibodyfragments, other binding polypeptides, peptides, and non-peptide smallmolecules, that bind to VEGF and are capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with VEGF activities.Thus, the term “VEGF activities” specifically includes VEGF mediatedbiological activities of VEGF.

The term “VEGF trap” as used herein means a protein, such as a fusionmolecule, that binds to VEGF and is capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with VEGF activities. Anexample of a VEGF trap is aflibercept.

The term “anti-VEGF antibody” or “an antibody that binds to VEGF” refersto an antibody that is capable of binding to VEGF with sufficientaffinity and specificity that the antibody is useful as a diagnosticand/or therapeutic agent in targeting VEGF. Anti-VEGF antibodiessuppress the growth of a variety of human tumor cell lines in nude mice(Kim et al., Nature 362:841-844 (1993); Warren et al., J. Clin. Invest.95:1789-1797 (1995); Borgström et al., Cancer Res. 56:4032-4039 (1996);Melnyk et al., Cancer Res. 56:921-924 (1996)) and also inhibitintraocular angiogenesis in models of ischemic retinal disorders. Adamiset al., Arch. Ophthalmol. 114:66-71 (1996). For example, the anti-VEGFantibody can be used as a therapeutic agent in targeting and interferingwith diseases or conditions wherein the VEGF activity is involved. See,e.g., U.S. Pat. Nos. 6,582,959, 6,703,020; WO98/45332; WO 96/30046;WO94/10202, WO2005/044853; EP 0666868B1; US Patent Applications20030206899, 20030190317, 20030203409, 20050112126, 20050186208, and20050112126; Popkov et al., Journal of Immunological Methods 288:149-164(2004); and WO2005012359. The antibody selected will normally have asufficiently strong binding affinity for VEGF. For example, the antibodymay bind hVEGF with a K_(d) value of between 100 nM-1 pM. Antibodyaffinities may be determined by a surface plasmon resonance based assay(such as the BIAcore assay as described in PCT Application PublicationNo. WO2005/012359); enzyme-linked immunoabsorbent assay (ELISA); andcompetition assays (e.g. RIA's), for example. The antibody may besubjected to other biological activity assays, e.g., in order toevaluate its effectiveness as a therapeutic. Such assays are known inthe art and depend on the target antigen and intended use for theantibody. Examples include the HUVEC inhibition assay; tumor cell growthinhibition assays (as described in WO 89/06692, for example);antibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonisticactivity or hematopoiesis assays (see WO 95/27062). An anti-VEGFantibody will usually not bind to other VEGF homologues such as VEGF-α,VEGF-C, VEGF-D or VEGF-E, nor other growth factors such as P1GF, PDGF orbFGF.

In one embodiment, anti-VEGF antibodies include a monoclonal antibodythat binds to the same epitope as the monoclonal anti-VEGF antibodyA4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanizedanti-VEGF monoclonal antibody (see Presta et al. (1997) Cancer Res.57:4593-4599), including but not limited to the antibody known as“bevacizumab” also known as “rhuMAb VEGF” or “AVASTIN®.” AVASTIN® ispresently commercially available. Nonlimiting exemplary cancers that maybe treated with bevacizumab include non-small cell lung cancer,colorectal cancer, breast cancer, renal cancer, ovarian cancer,glioblastoma multiforme, pediatric osteosarcoma, gastric cancer andpancreatic cancer. Bevacizumab comprises mutated human IgG₁ frameworkregions and antigen-binding complementarity-determining regions from themurine antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Bevacizumab and other humanized anti-VEGF antibodies arefurther described in U.S. Pat. Nos. 6,884,879, and 7,169,901. Additionalanti-VEGF antibodies are described in PCT Application Publication Nos.WO2005/012359 and WO2009/073160; U.S. Pat. Nos. 7,060,269, 6,582,959,6,703,020; 6,054,297; WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1;U.S. Patent Application Publication Nos. 2006009360, 20050186208,20030206899, 20030190317, 20030203409, and 20050112126; and Popkov etal., Journal of Immunological Methods 288:149-164 (2004).

A “effective amount” or “therapeutically effective amount” refers to anamount of a drug effective to treat a disease or disorder in a subject.In certain embodiments, an effective amount refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic or prophylactic result. A therapeutically effectiveamount of FGFR1 fusion protein of the invention may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the substance/molecule, agonist orantagonist to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the FGFR1 fusion proteins are outweighed by thetherapeutically beneficial effects. In the case of cancer, the effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and typically stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and typically stop) tumor metastasis; inhibit, to someextent, tumor growth; allow for treatment of the tumor, and/or relieveto some extent one or more of the symptoms associated with the disorder.To the extent the drug may prevent growth and/or kill existing cancercells, it may be cytostatic and/or cytotoxic.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Herein, “concurrent” dosing refers to the administration of twotherapeutic molecules within an eight hour time period. In someembodiments, two therapeutic molecules are administered at the sametime. Two therapeutic molecules are considered to be administered at thesame time (i.e. simultaneously) if at least a portion of a dose of eachtherapeutic molecule is administered within 1 hour. Two therapeuticmolecules are administered concurrently if at least one dose isadministered concurrently, even if one or more other doses are notadministered concurrently. In some embodiments, concurrentadministration includes a dosing regimen when the administration of oneor more therapeutic molecule(s) continues after discontinuing theadministration of one or more other therapeutic molecules(s).

Administration “in combination with” one or more further therapeuticagents includes concurrent (including simultaneous) and consecutive(i.e., sequential) administration in any order.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Pharmacokinetics of FP-1039 at doses from 0.5-16.0 mg/kg. Arrowsindicate days of FP-1039 administration. PK samples on Days 8 and 15 arepre-dose (trough), while detailed PK sampling is performed followingDose 1 (Day 1) and after 4 weekly doses (Day 22).

FIG. 2. Summary of plasma free FGF-2 levels at different timepointsacross all dosing cohorts (0.5-16 mg/kg). Free FGF-2 was measuredpre-dose Day 1, 24-hours post the 1st dose, pre-dose Day 8, Day 15, andDay 36. The Day 36 samples are two weeks following FP-1039 dosing.

FIG. 3. Free FGF-2 plasma levels in normal subjects and patients beforeand after a single dose of FP-1039. All patients had elevated FGF-2plasma levels compared to plasma from normal subjects. Plasma free-FGF-2levels in cancer patients treated with FP-1039 all decreased relative topre-dose levels (overall average decrease of 76%).

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The data provided here show for the first time that a soluble FibroblastGrowth Factor Receptor 1 (FGFR1)/Fc fusion protein, FP-1039, can beadministered safely to human patients at concentrations at about 0.5mg/kg body weight (i.e., up to at least about 16 mg/kg) and that suchconcentrations are well-tolerated. Accordingly, the present inventionprovides for administration of FP-1039 (i.e., SEQ ID NO:8) to humanindividuals, e.g., cancer patients, at concentrations from a dose ofabout 2 mg/kg body weight to at least about 30 mg/kg. In view of thesenew data, it is believed that other soluble FGFR1 fusion proteins canalso be safely administered to humans at these same elevatedconcentrations, for example, to treat cancer.

II. Methods of Treatment

The present invention provides methods of treating a human having acancer, the method comprising administering to the human a soluble FGFR1fusion protein (e.g., FP-1039) as described herein at a dose of about 2mg/kg body weight to about 30 mg/kg body weight.

In some embodiments, the human having a cancer has an FGF-2 plasmaconcentration above the average FGF-2 plasma concentration of the humanwithout cancer. In some embodiments, the human having a cancer has anFGF-2 plasma concentration of at least 6 pg/ml or at least 10 pg/mlprior to the administration of the soluble FGFR1 fusion protein. In someembodiments, as used herein, FGF-2 levels are determined as measured byan electrochemiluminescence (ECL) assay (Meso Scale Discovery,Gaithersburg, Md.), which utilizes an anti-FGF-2 antibody (Meso ScaleDiscovery) as the primary antibody and a ruthenium metal chelate(Sulfo-Tag) anti-human growth factor antibody blend as the secondaryantibody. Detection is accomplished using a reader such as MSD 512400reader (Meso Scale Discovery, Sector Image 2400, Model #1250). Fordetecting FGF-2 levels, the manufacturer's instructions are followed,with the following modification: the Assay Diluent GF1 is replaced withCalibrator Diluent GF1.

In another aspect, the method of treating the human having the cancercomprises administering soluble FGFR1 fusion protein (e.g., FP-1039) ina dose that results in sustained target engagement in the plasma evenafter seven days post-administration or longer. In some embodiments, thesoluble FGFR1 fusion protein is administered in a dose that results insustained engagement of the target FGF-2 in the plasma even after sevendays post-administration or longer. Thus, in some embodiments, thesoluble FGFR1 fusion protein is administered at a dose such that, atseven days after administration, the human has a free FGF-2 plasmaconcentration of less than 4 pg/ml. In some embodiments, the solubleFGFR1 fusion protein is administered at a dose such that, at seven daysafter administration, the human has a free FGF-2 plasma concentration ofless than 3 pg/ml. In some embodiments as described in this paragraph,the soluble FGFR1 fusion protein consists of or comprises SEQ ID NO:8.

A. Conditions Suitable for Treatment

The soluble FGFR1 fusion proteins of the present invention (includingbut not limited to FP-1039) find use in treating both metastatic andnon-metastatic forms of cancer, including but not limited to, pancreaticcancer, breast cancer, gastric cancer, bladder cancer, oral cancer,ovarian cancer, thyroid cancer, lung cancer, prostate cancer, uterinecancer, endometrial cancer, testicular cancer, neuroblastoma, squamouscell carcinoma of the head, neck, cervix and vagina, multiple myeloma,soft tissue and osteogenic sarcoma, colorectal cancer, liver cancer(i.e., hepatocarcinoma), renal cancer (i.e., renal cell carcinoma),mesothelioma, cervical cancer, anal cancer, bile duct cancer,gastrointestinal carcinoid tumors, gastrointestinal stromal tumors(GIST), esophageal cancer, laryngeal cancer, gall bladder cancer, smallintestine cancer, cancer of the central nervous system, skin cancer,choriocarcinoma; osteogenic sarcoma, rhabdomyosarcoma, fibrosarcoma,glioma, glioblastoma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma,Burkitt's lymphoma, Small Cell lymphoma, Large Cell lymphoma, monocyticleukemia, myelogenous leukemia, acute lymphocytic leukemia, and acutemyelocytic leukemia. In some embodiments, the cancer to be treated isprostate cancer, breast cancer, colorectal cancer, lung cancer,endometrial cancer, head and neck cancer, laryngeal cancer, livercancer, renal cancer, glioblastoma or pancreatic cancer.

In some embodiments, the humans treated with a soluble FGFR1 fusionprotein have a cancer that is characterized by a Fibroblast GrowthFactor Receptor 2 (FGFR2) with a ligand-dependent activating mutation. Aligand-dependent activating mutant is a FGFR2 variant whose biologicaleffects depend on the binding of FGFR2 to one or more of its ligands,such as a mutation that causes alterations in the ligand bindingproperties of FGFR2. An observed FGFR2 mutation in endometrial tumorcells is S252W, found in about 7% of cases. In addition, a P253Rmutation occurs in about 2% of endometrial cancer cases. These S252W andP253R mutations are the same mutations found in the germline of patientswith the genetic disease Apert Syndrome, which causes craniosynostosisand syndactyl). Structural and biochemical studies of the FGFR2 S252Wmutant receptor suggest that S252W and P253R mutants may cause the FGFR2protein to bind more tightly to its normal FGF ligands as well as tobind other FGF ligands to which it normally does not bind. See K. Yu etal., Proc. Natl. Acad. Sci. USA 97: 14536-41 (2000); O. A. Ibrahimi etal., Proc. Natl. Acad. Sci. USA 98: 7182-87 (2001).

In some embodiments, expression of an FGFR2 having a ligand-dependentactivating mutation is detected by biopsying the cancer and analyzingthe nucleic acid of the tumor cells of the cancer. Whether theligand-dependent activating mutation is detectably expressed in thenucleic acid can be analyzed using any method known in the art,including but not limited to polymerase chain reaction (PCR),quantitative PCR, RT-PCR, or sequencing analysis. In some embodiments,expression of an FGFR2 having a ligand-dependent activating mutation isdetected in tumor cells by PCR amplification and sequencing of exon 7 ofthe genomic FGFR2 gene using primer sequences described by Dutt et al.,PNAS105(25):8713-7 (2008).

B. Dosages, Formulations and Duration

The compositions of the present invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include, but are not limited to, theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. For the prevention or treatment ofdisease, the appropriate dosage of an FGFR1 fusion protein of theinvention (when used alone or in combination with one or more otheradditional therapeutic agents) will depend on the type of disease to betreated, the severity and course of the disease, whether the FGFR1fusion protein is administered for preventive or therapeutic purposes,the intended aggressiveness of the treatment regime, previous therapy,the patient's clinical history and response to the FGFR1 fusion protein,and the discretion of the attending physician.

The compositions for administration will commonly comprise a solubleFGFR1 fusion protein (i.e., an extracellular domain of an FGFR1polypeptide linked to a Fc polypeptide, such as but not limited toFP-1039) dissolved in a pharmaceutically acceptable carrier, e.g., anaqueous carrier. A variety of aqueous carriers can be used, e.g.,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of soluble FGFR1 fusion proteinin these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thepatient's needs.

Thus, a typical pharmaceutical composition for administration will varyaccording to the agent and method of administration (e.g. intravenous orsubcutaneous). Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

Pharmaceutical formulations for use with the present invention can beprepared by mixing a soluble FGFR1 fusion protein having the desireddegree of purity with optional pharmaceutically acceptable carriers,excipients or stabilizers. Such formulations can be lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsused. Acceptable carriers, excipients or stabilizers can be acetate,phosphate, citrate, and other organic acids; antioxidant (e.g., ascorbicacid); preservatives; low molecular weight polypeptides; proteins, suchas serum albumin or gelatin, or hydrophilic polymers such aspolyvinylpyllolidone; and amino acids, monosaccharides, disaccharides,and other carbohydrates including glucose, mannose, or dextrins;chelating agents; and ionic and non-ionic surfactants (e.g.,polysorbate); salt-forming counter-ions such as sodium; metal complexes(e.g., Zn-protein complexes); and/or non-ionic surfactants.

In some embodiments, the soluble FGFR1 fusion protein (including but notlimited to FP-1039) is administered at a dose of about 2 mg/kg bodyweight to about 30 mg/kg body weight. In some embodiments, the solubleFGFR1 fusion protein (including but not limited to FP-1039) isadministered at a dose of about 8 mg/kg body weight to about 20 mg/kgbody weight. In some embodiments, the soluble FGFR1 fusion protein(including but not limited to FP-1039) is administered at a dose ofabout 8 mg/kg body weight, about 10 mg/kg body weight, about 11 mg/kgbody weight, about 12 mg/kg body weight, about 13 mg/kg body weight,about 14 mg/kg body weight, about 15 mg/kg body weight, about 16 mg/kgbody weight, about 17 mg/kg body weight, about 18 mg/kg body weight,about 19 mg/kg body weight, about 20 mg/kg body weight, about 24 mg/kgbody weight, or about 30 mg/kg body weight, or in a dose ranging fromone to another of the above values. In some embodiments, dosages may beadministered twice a week, weekly, every other week, at a frequencybetween weekly and every other week, every three weeks, every fourweeks, or every month.

In certain embodiments, dosages of the soluble FGFR1 fusion protein canbe calculated in two ways depending on the extinction coefficient (EC)used. The extinction coefficient differs depending on whether theglycosylation of the protein is taken into account. In one embodiment,the extinction coefficient based on the amino acid composition ofFP-1039, for example, is 1.42 mL/mg*cm. In another embodiment, when thecarbohydrate portion as well as the protein portion of FP-1039 isaccounted for, the extinction coefficient is 1.11 mL/mg*cm. Calculationof the FP-1039 dose using an EC of 1.11 mL/mg*cm increases thecalculated dose by 28%, as shown in Table 1. Although the dosescalculated using the two extinction coefficients are different, themolar concentrations, or the actual amounts of drug administered, areidentical. Unless otherwise noted, the doses disclosed herein are eachcalculated using the extinction coefficient that does not take accountof glycosylation. For FP-1039, this extinction coefficient is 1.42mL/mg*cm. How these dosages compare to those calculated using theextinction coefficient that takes account of glycosylation is shown inTable 1. As can be seen from Table 1, a dosage of 8 mg/kg (e.g., 7.8 and8.0) using an EC of 1.42 mL/mg*cm herein corresponds to a dosage of 10mg/kg (e.g. 10.0 and 10.2) when calculated using an EC of 1.11 mL/mg*cm.A dosage of 16 mg/kg (e.g. 15.6 and 16.0 mg/kg) herein using an EC of1.42 mL/mg*cm corresponds to a dosage of about 20 mg/kg (e.g. 20.0 and20.5) when calculated using an EC of 1.11 mL/mg*cm. As noted in the“Definitions” section above, measured numbers provided herein areapproximate and encompass values having additional significant digitsthat are rounded off. For instance, 8 mg/kg encompasses values with twosignificant digits such as 7.6, 7.8, 8.0, 8.2, 8.4, and 8.45, each ofwhich round to 8. Likewise, a value such as 16 mg/kg encompasses valueswith three significant digits that round to 16, such as, for example15.6 and 16.0.

TABLE 1 Conversion of FP-1039 Dose Dose^(a) Dose^(a) EC = 1.42 mL/mg*cmEC = 1.11 mL/mg*cm 0.5 0.6 0.75 1.0 1.0 1.3 2.0 2.6 3.0 3.8 4.0 5.1 5.06.4 6.0 7.7 7.0 9.0 7.8 10.0 8.0 10.2 9.0 11.5 10.0 12.8 11.0 14.1 12.015.4 13.0 16.6 14.0 17.9 15.0 19.2 15.6 20.0 16.0 20.5 17.0 21.8 18.023.0 19.0 24.3 20.0 25.6 30.0 38.4 ^(a)Doses shown in mg/kg.

In some embodiments, the patient is treated with a combination of theFGFR1 fusion protein (e.g., FP-1039) and one or more other therapeuticagents(s). The combined administration includes coadministration orconcurrent administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein optionally there is a time period while both (or all)active agents simultaneously exert their biological activities. Theeffective amounts of therapeutic agents administered in combination withan FGFR1 fusion protein will be at the physician's or veterinarian'sdiscretion. Dosage administration and adjustment is done to achievemaximal management of the conditions to be treated. The dose willadditionally depend on such factors as the type of therapeutic agent tobe used and the specific patient being treated.

In some embodiments, the patient is treated with a combination of theFGFR1 fusion protein (e.g., FP-1039) and a VEGF antagonist. In someembodiments, the VEGF antagonist is a VEGF trap, such as aflibercept. Insome embodiments, the VEGF antagonist is a VEGF antibody. In someembodiments, the VEGF antibody is bevacizumab. One exemplary dosage ofbevacizumab would be in the range from about 0.05 mg/kg to about 20mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg,7.5 mg/kg, 10 mg/kg or 15 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g., every week, every two, or every three weeks.

In some embodiments, the FGFR1 fusion protein (e.g., FP-1039) isadministered in combination with another therapeutic agent, such aschemotherapeutic agent or anti-angiogenic agent, at the recommended orprescribed dosage and/or frequency of the therapeutic agent.

C. Methods of Administration

The soluble FGFR1 fusion proteins of the present invention areadministered to a human patient in accord with known methods, such asintravenous administration, e.g., as a bolus or by continuous infusionover a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes. The administration maybe local or systemic. In some embodiments, the soluble FGFR1 fusionproteins are administered intravenously. The methods described hereinare based on administering a soluble FGFR1 fusion protein to a humanpatient as a continuous infusion over a period of 30 minutes. However,the present invention also contemplates administering the soluble FGFR1fusion protein over a shorter or longer period of time, e.g., over aperiod of one hour.

The compositions comprising the FGFR1 fusion proteins of the inventioncan be administered as needed to subjects. In certain embodiments, aneffective dose of the FGFR1 fusion proteins of the invention isadministered to a subject one or more times. In various embodiments, aneffective dose of the FGFR1 fusion proteins of the invention isadministered to the subject at least once a month, at least twice amonth, once a week, twice a week, or three times a week. In variousembodiments, an effective dose of the FGFR1 fusion proteins of theinvention is administered to the subject for at least a week, at least amonth, at least three months, at least six months, or at least a year.

D. Combination Therapy

The FGFR1 fusion proteins of the present invention may be administeredalone or with other modes of treatment. They may be provided before,substantially contemporaneous with, or after other modes of treatment,for example, surgery, chemotherapy, radiation therapy, or theadministration of other therapeutics, including for example, smallmolecules and other biologics, such as a therapeutic antibody.

In some embodiments, an FGFR1 fusion protein of the present invention isthe only therapeutically active agent administered to a patient. Incertain embodiments, the FGFR1 fusion protein is administered incombination with one or more other therapeutic agents, including but notlimited to anti-angiogenic agents, chemotherapeutic agents, cytokines,growth inhibitory agents, anti-hormonal agents, kinase inhibitors,cytotoxic agents, cardioprotectants, or other therapeutic agents. TheFGFR1 fusion protein may be administered concurrently with one or moreother therapeutic regimens. In some embodiments, the FGFR1 fusionprotein may be administered in combination with one or more antibodies.

Anti-angiogenic therapy in relationship to cancer is a cancer treatmentstrategy aimed at inhibiting the development of tumor blood vesselsrequired for providing nutrients to support tumor growth. In someembodiments, because angiogenesis is involved in both primary tumorgrowth and metastasis, the anti-angiogenic treatment provided by theinvention is capable of inhibiting the neoplastic growth of tumor at theprimary site as well as preventing metastasis of tumors at the secondarysites, therefore allowing attack of the tumors by other therapeutics. Inone embodiment of the invention, anti-cancer agent or therapeutic is ananti-angiogenic agent. In another embodiment, anti-cancer agent is achemotherapeutic agent.

Many anti-angiogenic agents have been identified and are known in thearts, including those listed herein, e.g., listed under Definitions, andby, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al.,Nature Reviews: Drug Discovery, 3:391-400 (2004); and Sato Int. J. Clin.Oncol., 8:200-206 (2003). See also, US Patent Application US20030055006.In some embodiments, two or more angiogenic inhibitors may optionally beco-administered to the patient in addition to an FGFR1 fusion protein ofthe invention.

In some embodiments, other therapeutic agents that may be combined withthe FGFR1 fusion protein are VEGF antagonists or VEGF receptorantagonists. In some embodiments, other therapeutic agents useful forcombination tumor therapy with the FGFR1 fusion protein includeantagonists of other factors that are involved in tumor growth, such asEGFR, ErbB2 (also known as Her2) ErbB3, ErbB4, or TNF. In someembodiments, the FGFR1 fusion protein can be used in combination withsmall molecule receptor tyrosine kinase inhibitors (RTKIs) that targetone or more tyrosine kinase receptors such as VEGF receptors, FGFreceptors, EGF receptors and PDGF receptors. Many therapeutic smallmolecule RTKIs are known in the art, including, but are not limited to,vatalanib (PTK787), erlotinib (TARCEVA®), OSI-7904, ZD6474 (ZACTIMA®),ZD6126 (ANG453), ZD1839, sunitinib (SUTENT®), semaxanib (SU5416),AMG706, AG013736, Imatinib (GLEEVEC®), MLN-518, CEP-701, PKC-412,Lapatinib (GSK572016), VELCADE®, AZD2171, sorafenib (NEXAVAR®), XL880,and CHIR-265.

In some embodiments, the FGFR1 fusion protein and the one or more othertherapeutic agents can be administered concurrently, simultaneously, orsequentially in an amount and for a time sufficient to reduce oreliminate the occurrence or recurrence of a tumor, a dormant tumor, or amicrometastases. The FGFR1 fusion protein and the one or more othertherapeutic agents can be administered as maintenance therapy to preventor reduce the likelihood of recurrence of the tumor.

III. Soluble FGFR1Fusion Proteins

A. FGFR1 ECD Fusion Molecules

Soluble FGFR1 fusion proteins or FGFR1 ECD fusion molecules, refer toproteins comprising an FGFR1 ECD polypeptide linked to at least onefusion partner, wherein the soluble FGFR1 fusion protein lacks atransmembrane domain (e.g., an FGFR1 transmembrane domain) and is notbound to the cellular membrane. The fusion partner may be joined toeither the N-terminus or the C-terminus of the FGFR1 ECD polypeptide.When the fusion partner is a polypeptide, the FGFR1 ECD may be joined toeither the N-terminus or the C-terminus of the fusion partner.

B. FGFR1 ECDs

FGFR1 ECD molecules are provided. In some embodiments, FGFR1 ECDsconsist of native FGFR1 ECDs, FGFR1 ECD variants, FGFR1 ECDs comprisingan Ig domain III chosen from IIIb and Inc, native FGFR1-IIIb ECD, nativeFGFR1-IIIc ECD, FGFR1-IIIb ECD variants, FGFR1-IIIc ECD variants, FGFR1ECD fragments, native FGFR1 ECD fragments, variants of FGFR1 ECDfragments, FGFR1 ECD glycosylation mutants, and FGFR1 ECD fusionmolecules, as well as non-human FGFR1 ECDs. All FGFR1 ECDs are able tobind FGF-2. In some embodiments, the FGFR1 ECD includes a signalpeptide, either from FGFR1, or from another FGFR, or from anotherprotein. In other embodiments, no signal peptide is included.

The FGFR1 ECD proteins of the invention can comprise an entire FGFR1ECD, including that of wildtype FGFR1-IIIb or wildtype FGFR1-IIIc ECD,or for example a variant or fragment of the FGFR1 ECD (e.g., a variantor fragment having at least 95% amino acid sequence identity to awildtype FGFR1 ECD) that retains the ability to bind FGF-2. In someembodiments, a variant of the native FGFR1 ECD, for example, lacking thefirst immunoglobulin domain is provided. See, e.g., U.S. Pat. No.6,384,191. In some embodiments, a variant of the native FGFR1 ECD havinga deletion of one or more and up to 22 amino acid residues counting fromthe C-terminus of the native FGFR1 ECD of SEQ ID NO:1, is provided. Insome embodiments, the FGFR1 ECD has the final 22 amino acids of theC-terminus deleted, while in others, the FGFR1 ECD has the final 19, 14,9, 8, or 4 C-terminal amino acids deleted in comparison to SEQ ID NO:1.See, e.g., SEQ ID NOs:2-6.

In some embodiments, the FGFR1 ECD of the present invention has at least90%, at least 92%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or higher amino acid sequence identity to a wildtypeFGFR1-IIIc ECD (SEQ ID NO:1). In some embodiments, the FGFR1 ECD of thepresent invention has at least 90%, at least 92%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or higher amino acidsequence identity to a wildtype FGFR1-IIIc ECD (SEQ ID NO:1) and has theability to bind FGF-2.

Non-limiting exemplary FGFR1 ECD fragments include human FGFR1 ECDending at amino acid 339 (counting from the first amino acid of themature form, without the signal peptide). In some embodiments, an FGFR1ECD fragment ends at an amino acid between amino acid 339 and amino acid360 (counting from the first amino acid of the mature form, without thesignal peptide).

Examples of FGFR1 ECD fragments include those having the C-terminalamino acid residues LYLE (SEQ ID NO:9) or MTSPLYLE (SEQ ID NO:10) orVMTSPLYLE (SEQ ID NO:11) or AVMTSPLYLE (SEQ ID NO:12) or EERPAVMTSPLYLE(SEQ ID NO:13) or LEERPAVMTSPLYLE (SEQ ID NO:14) or ALEERPAVMTSPLYLE(SEQ ID NO:15) deleted as compared to either a native FGFR1-IIIb orFGFR1-IIIc. Further examples include those having the C-terminal aminoacid residues KALEERPAVMTSPLYLE (SEQ ID NO:16) or RPVAKALEERPAVMTSPLYLE(SEQ ID NO:17) deleted as compared to a native FGFR1-IIIb orEALEERPAVMTSPLYLE (SEQ ID NO:18) deleted as compared to a nativeFGFR1-IIIc. Point mutations, truncations, or internal deletions orinsertions within the ECD amino acid sequence may be made within theFGFR1 ECD so long as FGF-2 binding activity is retained.

C. Fusion Partners and Conjugates

In certain embodiments, a fusion partner is selected that impartsfavorable pharmacokinetics and/or pharmacodynamics on the FGFR1 ECDprotein. For example, in certain embodiments, a fusion partner isselected that increases the half-life of the FGFR1 ECD fusion moleculerelative to the corresponding FGFR1 ECD without the fusion partner. Byincreasing the half-life of the molecule, a lower dose and/orless-frequent dosing regimen may be required in therapeutic treatment.Further, the resulting decreased fluctuation in FGFR1 ECD serum levelsmay improve the safety and tolerability of the FGFR1 ECD-basedtherapeutics.

Many different types of fusion partners are known in the art. Oneskilled in the art can select a suitable fusion partner according to theintended use. Non-limiting exemplary fusion partners include polymers,polypeptides, lipophilic moieties, and succinyl groups. Exemplarypolypeptide fusion partners include serum albumin (e.g. human serumalbumin or HSA) and an antibody Fc domain. Exemplary polymer fusionpartners include, but are not limited to, polyethylene glycol, includingpolyethylene glycols having branched and/or linear chains.

D. Oligomerization Domain Fusion Partners

In various embodiments, oligomerization offers certain functionaladvantages to a fusion protein, including, but not limited to,multivalency, increased binding strength, and the combined function ofdifferent domains. Accordingly, in certain embodiments, a fusion partnercomprises an oligomerization domain, for example, a dimerization domain.Exemplary oligomerization domains include, but are not limited to,coiled-coil domains, including alpha-helical coiled-coil domains;collagen domains; collagen-like domains, and certain immunoglobulindomains. Certain exemplary coiled-coil polypeptide fusion partnersinclude the tetranectin coiled-coil domain; the coiled-coil domain ofcartilage oligomeric matrix protein; angiopoietin coiled-coil domains;and leucine zipper domains. Certain exemplary collagen or collagen-likeoligomerization domains include, but are not limited to, those found incollagens, mannose binding lectin, lung surfactant proteins A and D,adiponectin, ficolin, conglutinin, macrophage scavenger receptor, andemilin.

E. Antibody Fc Immunoglobulin Domain Fusion Partners

Many Fc domains that could be used as fusion partners are known in theart. One skilled in the art can select an appropriate Fc domain fusionpartner according to the intended use. In certain embodiments, a fusionpartner is an Fc immunoglobulin domain. An Fc fusion partner may be awildtype Fc found in a naturally occurring antibody, a variant thereof,or a fragment thereof. Non-limiting exemplary Fc fusion partners includeFcs comprising a hinge and the CH2 and CH3 constant domains of a humanIgG, for example, human IgG1, IgG2, IgG3, or IgG4. In some embodiments,the Fc fusion partner does not include an Fc hinge region. Certainadditional Fc fusion partners include, but are not limited to, human IgAand IgM. In certain embodiments, an Fc fusion partner comprises a C237Smutation. In certain embodiments, an Fc fusion partner comprises ahinge, CH2, and CH3 domains of human IgG2 with a P331S mutation, asdescribed in U.S. Pat. No. 6,900,292.

F. Albumin Fusion Partners and Albumin-Binding Molecule Fusion Partners

In certain embodiments, a fusion partner is an albumin. Certainexemplary albumins include, but are not limited to, human serum albumin(HSA) and fragments of HSA that are capable of increasing the serumhalf-life and/or bioavailability of the polypeptide to which they arefused. In certain embodiments, a fusion partner is an albumin-bindingmolecule, such as, for example, a peptide that binds albumin or amolecule that conjugates with a lipid or other molecule that bindsalbumin. In certain embodiments, a fusion molecule comprising HSA isprepared as described, e.g., in U.S. Pat. No. 6,686,179.

G. Polymer Fusion Partners

In certain embodiments, a fusion partner is a polymer, for example,polyethylene glycol (PEG). PEG may comprise branched and/or linearchains. In certain embodiments, a fusion partner comprises achemically-derivatized polypeptide having at least one PEG moietyattached. Pegylation of a polypeptide may be carried out by any methodknown in the art. One skilled in the art can select an appropriatemethod of pegylating a particular polypeptide, taking into considerationthe intended use of the polypeptide. Certain exemplary PEG attachmentmethods include, for example, EP 0 401 384; Malik et al., Exp. Hematol.,20:1028-1035 (1992); Francis, Focus on Growth Factors, 3:4-10 (1992); EP0 154 316; EP 0 401 384; WO 92/16221; and WO 95/34326. As non-limitingexamples, pegylation may be performed via an acylation reaction or analkylation reaction, resulting in attachment of one or more PEG moietiesvia acyl or alkyl groups. In certain embodiments, PEG moieties areattached to a polypeptide through the α- or ε-amino group of one or moreamino acids, although any other points of attachment known in the artare also contemplated.

Pegylation by acylation typically involves reacting an activated esterderivative of a PEG moiety with a polypeptide. A non-limiting exemplaryactivated PEG ester is PEG esterified to N-hydroxysuccinimide (NHS). Asused herein, acylation is contemplated to include, without limitation,the following types of linkages between a polypeptide and PEG: amide,carbamate, and urethane. See, e.g., Chamow, Bioconjugate Chem.,5:133-140 (1994). Pegylation by alkylation typically involves reacting aterminal aldehyde derivative of a PEG moiety with a polypeptide in thepresence of a reducing agent. Non-limiting exemplary reactive PEGaldehydes include PEG propionaldehyde, which is water stable, and monoC1-C10 alkoxy or aryloxy derivatives thereof. See, e.g., U.S. Pat. No.5,252,714.

In certain embodiments, a pegylation reaction results in poly-pegylatedpolypeptides. In certain embodiments, a pegylation reaction results inmono-, di-, and/or tri-pegylated polypeptides. Further, desiredpegylated species may be separated from a mixture containing otherpegylated species and/or unreacted starting materials using variouspurification techniques known in the art, including among others,dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gelfiltration chromatography, and electrophoresis.

H. Exemplary Attachment of Fusion Partners

The fusion partner may be attached, either covalently or non-covalently,to the amino-terminus or the carboxy-terminus of the FGFR1 ECD. Theattachment may also occur at a location within the FGFR1 ECD other thanthe amino-terminus or the carboxy-terminus, for example, through anamino acid side chain (such as, for example, the side chain of cysteine,lysine, histidine, serine, or threonine).

In either covalent or non-covalent attachment embodiments, a linker maybe included between the fusion partner and the FGFR1 ECD. Such linkersmay be comprised of amino acids and/or chemical moieties. Exemplarymethods of covalently attaching a fusion partner to an FGFR1 ECDinclude, but are not limited to, translation of the fusion partner andthe FGFR1 ECD as a single amino acid sequence and chemical attachment ofthe fusion partner to the FGFR1 ECD. When the fusion partner and theFGFR1 ECD are translated as single amino acid sequence, additional aminoacids may be included between the fusion partner and the FGFR1 ECD as alinker. In certain embodiments, the linker is selected based on thepolynucleotide sequence that encodes it, to facilitate cloning thefusion partner and/or FGFR1 ECD into a single expression construct (forexample, a polynucleotide containing a particular restriction site maybe placed between the polynucleotide encoding the fusion partner and thepolynucleotide encoding the FGFR1 ECD, wherein the polynucleotidecontaining the restriction site encodes a short amino acid linkersequence).

When the fusion partner and the FGFR1 ECD are covalently coupled bychemical means, linkers of various sizes can typically be includedduring the coupling reaction. Several methods of covalent coupling of apolypeptide to another molecule (i.e. fusion partner) are known. Thepolypeptide and fusion partner can also be non-covalently coupled.Exemplary methods of non-covalently attaching a fusion partner to anFGFR1 ECD include, but are not limited to, attachment through a bindingpair. Exemplary binding pairs include, but are not limited to, biotinand avidin or streptavidin, an antibody and its antigen, etc.

I. Polynucleotides Encoding FGFR1 Fusion Proteins

Nucleic acid molecules encoding FGFR1 ECDs and/or soluble FGFR1 fusionproteins can be synthesized by chemical methods or prepared bytechniques well known in the art. See, for example, Sambrook, et al.,Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989).

In some embodiments, a polynucleotide encoding a polypeptide of theinvention comprises a nucleotide sequence that encodes a signal peptide,which, when translated, will be fused to the amino-terminus of the FGFR1polypeptide. In other embodiments, the nucleotide sequence does notinclude a sequence encoding a signal peptide. As discussed above, thesignal peptide may be the native signal peptide, the signal peptide ofFGFR1, FGFR2, FGFR3, or FGFR4, or may be another heterologous signalpeptide. Exemplary signal peptides are known in the art, and aredescribed, e.g., in PCT Publication No. WO 2006/081430.

In some embodiments, the nucleic acid molecule comprising thepolynucleotide encoding the soluble FGFR1 fusion protein is anexpression vector that is suitable for expression in a selected hostcell. In some embodiments, the polynucleotide encoding the FGFR1 ECDpolypeptide (e.g., the polynucleotide encoding the polypeptide of any ofSEQ ID NOs:1-6) is inserted into the expression vector at a linker site,and the polynucleotide encoding the fusion partner polypeptide (e.g.,the polynucleotide encoding the Fc polypeptide of human IgG1) isinserted at a site following the FGFR1 ECD such that the FGFR1 ECD andFc components are in-frame when the nucleic acid molecule is transcribedand translated.

J. Production and Purification of FGFR1 Fusion Proteins

Cell lines and methods of producing soluble FGFR1 fusion proteins aredescribed in U.S. Pat. No. 7,678,890.

The FGFR1 fusion proteins of the present invention may be expressed froma vector in a host cell. In some embodiments, a vector is selected thatis optimized for expression of polypeptides in CHO-S or CHO-S-derivedcells. Exemplary such vectors are described, e.g., in Running Deer etal., Biotechnol. Prog. 20:880-889 (2004). In some embodiments, a vectoris chosen for in vivo expression of the polypeptides of the invention inanimals, including humans. In some embodiments, expression of thepolypeptide is under the control of a promoter that functions in atissue-specific manner.

Suitable host cells for expression of the FGFR1 fusion proteins of thepresent invention include, for example, prokaryotic cells, such asbacterial cells; or eukaryotic cells, such as fungal cells, plant cells,insect cells, and mammalian cells. Exemplary eukaryotic cells that canbe used to express polypeptides include, but are not limited to, Coscells, including Cos 7 cells; 293 cells, including 293-6E and 293-Tcells; CHO cells, including CHO-S and DG44 cells; and NS0 cells.

Introduction of a nucleic acid vector into a desired host cell can beaccomplished by any method known in the art, including, but not limitedto, calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, etc. Certain exemplary methods are described, e.g., inSambrook et al., Molecular Cloning, A Laboratory Manual, 3^(rd) ed. ColdSpring Harbor Laboratory Press (2001). Nucleic acids may be transientlyor stably transfected in the desired host cells, according to methodsknown in the art.

In some embodiments, a polypeptide can be produced in vivo in an animalthat has been engineered or transfected with a nucleic acid moleculeencoding the polypeptide, according to methods known in the art.

The FGFR1 fusion proteins of the present invention can be purified byvarious methods known in the art. Such methods include, but are notlimited to, the use of affinity matrices, ion exchange chromatography,and/or hydrophobic interaction chromatography. Suitable affinity ligandsinclude any ligands of the FGFR1 ECD or of the fusion partner, orantibodies thereto. For example, in the case of a fusion protein, aProtein A, Protein G, Protein A/G, or an antibody affinity column may beused to bind to an Fc fusion partner to purify a polypeptide of theinvention. Antibodies to the polypeptides of the invention may also beused to purify the polypeptides of the invention. Hydrophobicinteractive chromatography, for example, a butyl or phenyl column, mayalso suitable for purifying certain polypeptides. Many methods ofpurifying polypeptides are known in the art

Methods of constructing DNA coding sequences, vectors, and host cellsfor FGFR1 ECDs, as well as methods of expressing and purifying FGFR1ECDs are also described, for example, in WO2007/014123 and in U.S.patent application Ser. No. 12/535,479 and PCT ApplicationPCT/US09/52704, each filed Aug. 4, 2009.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. It is understood that various other embodiments maybe practiced, given the general description provided above.

Example 1 Dose-Finding Study to Evaluate the Safety and Tolerability ofthe FGFR1-Fc Fusion Protein FP-1039 in Patients with AdvancedMalignancies

FP-1039 (SEQ ID NO:8) is a highly glycosylated, dimerized, solublefusion protein consisting of a truncated extracellular domain of humanFGFR1 linked to the Fc region of human IgG₁. In preclinical studies,FP-1039 demonstrated significant anti-tumor activity in a variety ofdifferent xenograft models; enhanced anti-tumor activity when combinedwith cytotoxic or targeted anti-cancer drugs; and inhibited both FGF—and VEGF-mediated angiogenesis.

A Phase I clinical trial with FP-1039 had dosing cohorts at 0.5 mg/kgbody weight through 16 mg/kg body weight. Inclusion criteria includedsubjects having histologically or cytologically proven metastatic orlocally advanced, unresectable solid tumors for which standard curativeor palliative measures do not exist or are no longer effective; EasternCooperative Oncology Group (ECOG) Performance Status grade 0-2 (see Okenet al., Am. J. Clin. Oncol. 5:649-655 (1982), incorporated by referenceherein in its entirety); and washout of prior anti-cancer therapy.

FP-1039 was administered to subjects intravenously over 30 minutes oncea week for 4 infusions, followed by a two-week observation period. Basedon the observation of two dose-limiting toxicities (DLTs) in the threesubjects dosed at 1 mg/kg (one episode of bowel perforation and sepsisand one episode of grade 3 neutropenia), doses of 0.5 mg/kg and 0.75mg/kg were explored. Six subjects were dosed at 0.5 mg/kg and sixsubjects were dosed at 0.75 mg/kg. As shown in Table 3 below, adverseeffects included one adverse effect was observed at 0.5 mg/kg (grade 1erythema) and one adverse effect was observed at 0.75 mg/kg (grade 2urticaria). Other adverse events were observed but were not responsiblefor removal of subjects from treatment due to the adverse effects.

Although protocol-defined DLTs were observed in patients at 1 mg/kg and0.75 mg/kg doses, dose-escalation was continued beyond 1 mg/kg, andcohorts of subjects were tested at FP-1039 dose levels of 2.0 mg/kg, 4.0mg/kg, 8.0 mg/kg, and 16.0 mg/kg. In total, 33 subjects have been dosedto date (including those subjects dosed as described above). The tumortypes for the subjects enrolled in the clinical trial are shown in Table2.

TABLE 2 Cohorts and Dispositions of Subjects Treated Tumor Type Numberof Subjects (n = 33) Prostate cancer 5 Breast cancer 7 ^(a) Sarcoma 6^(b) Hepatobiliary 2 ^(c) Colorectal cancer 4 Lung cancer 3 Endometrialcancer 1 Laryngeal cancer 1 ^(d) Carcinoma of salivary gland 2 Carcinoid1 Pancreatic cancer 1 ^(a) Combines: liposarcoma, sarcoma of leg,leiomyosarcoma, chondrosarcoma, and mixed Mullerian Duct sarcoma ^(b)Combines: hepatocellular carcinoma and cholangiosarcoma ^(c) Combines:adenocarcinoma of colon and rectal carcinoma and adenocarcinoma of smallbowel ^(d) Combines: cancer of salivary gland and adenoid cysticcarcinoma

Safety

For each cohort, FP-1039 was administered intravenously over 30 minutesonce a week for 4 infusions, followed by a two-week observation period.Safety of the dosing level was evaluated by assessing for adverse eventsat each visit. Adverse Events were graded by the Common TerminologyCriteria for Adverse Events v3.0 (CTCAE). DLT was defined as anyFP-1039-associated Adverse Event of CTCAE grade 3 or higher. Aftercompletion of the initial treatment and observation period, subjectswith no evidence of disease progression or DLT after 4 infusions wereeligible to receive additional weekly infusions of FP-1039. The cohortsof subjects and dispositions or best responses for each cohort are shownin Table 3.

TABLE 3 Cohorts and Dispositions of Subjects Treated Original FP-1039Dose Level (mg/kg) Number of Subjects Disposition/Best Response ^(a)16.0 4 3 SD, 1 PD 8.0 5 4 PD, 1 SD, 4.0 3 3 SD 2.0 3 3 PD 1.0 6 2 SD, 3PD, 1 SAE ^(b) 0.75 6 2 SD, 1 AE ^(d), 3 PD 0.5 6 3 SD, 1 AE ^(c), 2 PDAE: Adverse Event; PD: Progressive Disease; SAE: Serious Adverse Event;SD: Stable Disease ^(a) Column indicates best response or if subjectswithdrew from treatment due to AEs ^(b) Bowel perforation and sepsis inpatient with tumor in bowel wall ^(c) Grade 1 erythema ^(d) Grade 2urticaria in patient with history of allergies

Generally, FP-1039 was well-tolerated without observations ofdrug-related weight loss, hypertension, or soft tissue calcification atdoses up through 16 mg/kg. No DLTs were observed in the 15 subjectsdosed from 2 mg/kg to 16 mg/kg.

Pharmacokinetics

The pharmacokinetics of FP1-1039 dosing cohorts (0.5 mg/kg, 0.75 mg/kg,1 mg/kg, 2 mg/kg, 4 mg/kg, and 8 mg/kg) was analyzed at multipletimepoints. Plasma samples were drawn at various times after the firstinfusion, on Days 8, 15, and 22 immediately following dosing, and atvarious times following dosing on Day 22. For sample collectiongenerally, whole blood was collected in K₂EDTA tubes, processed intoplasma, aliquotted into volumes of 0.5 ml and stored at ≦70° C.Collected samples were subsequently shipped to Prevalere Life Sciences(Rome, N.Y.) on dry ice. Separate aliquots were prepared for analysis ofeach parameter discussed below (pharmacokinetics, pharmacodynamics,anti-drug antibody, and neutralizing antibody). Samples received atPrevalere were kept at ≦70° C. until analysis.

FP-1039 concentration was measured in plasma from the subjects using aquantitative enzyme immunoassay methodology that measures free, activeFP-1039 in K₂EDTA plasma. Briefly, samples and controls were diluted inassay diluent containing excess heparin and loaded onto platespre-coated with recombinant human FGF-2 and pre-blocked. After a 1-hourincubation, the plate was washed and an anti-human IgG-Fc horseradishperoxidase (HRP) antibody added to detect bound FP-1039 using standardcolorimetric ELISA detection. The method was validated in accordancewith bioanalytical method validation guidelines and following GoodLaboratory Practices (GLP) prior to clinical sample testing at PrevalereLife Sciences.

Plasma concentrations vs. time curves were generated. Non-compartmentalanalysis was performed with WinNonlin professional version 5.2.1(Pharsight Corporation, Mountain View, Calif.) or PK Solutions 2.0™software (Version 2.0.6 for Windows, Excel 2002 edition; Summit ResearchServices, Montrose, Colo.). The area under the curve from time zero tothe last measureable time point (AUC_(0-t)) was estimated using thetrapezoid method. Linear regression over the last three or more timepoints was used to estimate the elimination rate constant (λ) which wasused to estimate terminal half-life (t_(1/2)) and AUC from zero toinfinity (AUC_(0-∞)) from the following equations:

t _(1/2)=ln(2)/λ

AUC_(0-∞)=AUC_(0-t)+C_(t)/λ

where C_(t) is the last measureable concentration. Plasma clearance (CL)was determined from the following equation:

CL=Dose/AUC_(0-∞).

The maximum concentration (C_(max)) and the time from the start ofinfusion of the maximum concentration (T_(max)) was determined directlyfrom the data.

As shown in FIG. 1, the mean plasma concentration for each dose cohortalong with standard deviation data was plotted against time. Thepharmacokinetic profile of FP-1039 was typical of a large protein withan initial distribution phase and a second (terminal/elimination) phase.As shown in FIG. 1, there was a linear, dose-dependent increase in drugexposure in the plasma compartment. At a dose of 8 mg/kg body weight,the individual subject terminal half-life (t_(1/2)) ranged from 113 to120 hours, with the mean of 117 hours (4.9 days) after the fourth doseon Day 22. The pharmacokinetic parameters C_(max), C_(min), and AUCincreased in proportion to the dose. As shown in FIG. 1, the plasmaconcentration of FP-1039 remains above 10 μg/ml even at one week postdosing. Furthermore, there was accumulation of FP-1039 in the plasmawhen comparing the C_(max), C_(min), and AUC data between the Day 1 andDay 22 doses (first vs. fourth dose). In summary, the pharmacokineticprofile of FP-1039 supports twice weekly, weekly, or less frequentdosing.

Pharmacodynamics

The target engagement of FGF-2 at various doses of FP-1039 was measuredat multiple timepoints. Plasma samples were drawn pre-dose (Day 1), 24hours post first dose (Day 2), and at Day 36 (14 days past 4^(th)/lastdose).

Plasma free FGF-2 levels were measured using a modified commercialimmunoassay kit (Meso Scale Discovery, MSD) which utilizes anelectrochemiluminescent (ECL) technology based on paired antibodies fordetection of FGF-2. The detection system employs a ruthenium metalchelate (Sulfo-Tag) antibody as the ECL label. The relative mass valuesfor natural FGF-2 in the plasma or serum samples is determined using therecombinant protein standards provided in the kit. Briefly, samples andcontrols were diluted in assay diluent and loaded onto plates pre-coatedwith antibodies against FGF-2 and pre-blocked. After a 2-hourincubation, the plate was washed and a detection antibody (SULFO-TAGanti-human growth factor detection antibody blend) was added. After a2-hour incubation, the plate was washed and read with the MSD SI2400reader (Meso Scale Discovery, Sector Image 2400, Model #1250) and thedata analyzed.

FGF-2 levels: Prior to FP-1039 treatment, clinical subjects had elevatedmean FGF-2 plasma concentrations relative to normal donors. As shown inFIGS. 2-3, treatment with FP-1039 results in a decrease in free plasmaFGF-2, suggesting that FP-1039 sequesters FGF-2 present in the blood.FIG. 2 shows that within 48 hours post dosing with FP-1039, there is asignificant decrease in plasma FGF-2 levels. Among all of the dosinglevels shown in FIGS. 2-3, plasma free FGF-2 levels in cancer patientsdecreased relative to pre-dose levels (overall average decrease of 76%).This data demonstrates that FP-1039 exhibits a high degree of targetengagement in plasma. Moreover, target engagement is maintainedthroughout the dosing schedule of weekly dosing. The data furthersuggest that target engagement may be maintained even after 2 weeks,supporting less frequent dosing.

Immune Response

To evaluate whether patients developed an immune response to FP-1039,samples were taken on Day 1, Day 15, Day 36, and every 3 monthsfollowing the initial dose. Anti-drug antibodies directed againstFP-1039 in the subject plasma samples were analyzed by anelectrochemiluminescent immunoassay (ECLA) utilizing Meso ScaleDiscovery (MSD) technology, which employs a ruthenium metal chelate(Sulfo-Tag) as the ECL label.

Preliminary analysis of anti-drug antibodies demonstrated thatapproximately 33% of subjects had transient, low titer antibodies at Day15. There was no relationship of ADAs to PK, target engagement, orsafety observations.

INFORMAL SEQUENCE LISTING Human FGFR1 isoform IIIc extracellular domainRPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKETDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL EERPAVMTSP LYLEHuman FGFR1 isoform IIIc extracellular domain Δ4 SEQ ID NO: 2RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKETDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL EERPAVMTSPHuman FGFR1 isoform IIIc extracellular domain Δ8 SEQ ID NO: 3RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKETDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL EERPAVHuman FGFR1 isoform IIIc extracellular domain Δ9 SEQ ID NO: 4RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKETDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL EERPAHuman FGFR1 isoform IIIc extracellular domain Δ14 SEQ ID NO: 5RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKETDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEALHuman FGFR1 isoform IIIc extracellular domain Δ19 SEQ ID NO: 6RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTRITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKETDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHRIGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLPANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKEMEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTEngineered human IgG1 Fc domain with C237S mutation SEQ ID NO: 7EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKHuman FGFR1 isoform IIIc extracellular domain Δ14 linked tohuman IgG1 Fc domain SEQ ID NO: 8RPSPTLPEQAQPWGAPVEVESFLVHPGDLLQLRCRLRDDVQSINWLRDGVQLAESNRTRITGEEVEVQDSVPADSGLYACVTSSPSGSDTTYFSVNVSDALPSSEDDDDDDDSSSEEKETDNTKPNPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKGNYTCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of treating a human having a cancer, wherein the human has afibroblast growth factor-2 (FGF-2) plasma concentration of at least 6pg/ml, the method comprising: administering to the human a solubleFibroblast Growth Factor Receptor 1 (FGFR1) fusion protein at a dose ofat least about 2 mg/kg body weight, wherein the fusion protein comprisesan extracellular domain of an FGFR1 polypeptide linked to a Fcpolypeptide.
 2. The method of claim 1, wherein the FGFR1 extracellulardomain comprises the amino acid sequence of SEQ ID NO:5.
 3. The methodof claim 1, wherein the soluble FGFR1 fusion protein comprises the aminoacid sequence of SEQ ID NO:8.
 4. The method of claim 1, wherein thesoluble FGFR1 fusion protein is administered at a dose of about 2 mg/kgbody weight to about 20 mg/kg body weight.
 5. The method of claim 4,wherein the soluble FGFR1 fusion protein is administered at a dose ofabout 8 mg/kg body weight to about 16 mg/kg body weight.
 6. The methodof claim 5, wherein the soluble FGFR1 fusion protein is administered ata dose of about 8 mg/kg body weight.
 7. The method of claim 5, whereinthe soluble FGFR1 fusion protein is administered at a dose of about 16mg/kg body weight.
 8. The method of claim 1, wherein the cancer isprostate cancer, breast cancer, colorectal cancer, lung cancer,endometrial cancer, head and neck cancer, laryngeal cancer, livercancer, renal cancer, glioblastoma, or pancreatic cancer.
 9. The methodof claim 1, wherein the human has an FGF-2 plasma concentration of atleast 10 pg/ml prior to the administration of the soluble FGFR1 fusionprotein.
 10. The method of claim 1, wherein the soluble FGFR1 fusionprotein is administered in combination with a chemotherapeutic agent ora VEGF antagonist.
 11. A method of treating a human having a cancer,wherein the cancer is characterized by a Fibroblast Growth FactorReceptor 2 (FGFR2) having a ligand-dependent activating mutation, themethod comprising: administering to the human a soluble FibroblastGrowth Factor Receptor 1 (FGFR1) fusion protein at a dose of at leastabout 2 mg/kg body weight, wherein the fusion protein comprises anextracellular domain of an FGFR1 polypeptide linked to a Fc polypeptide.12. The method of claim 11, wherein the FGFR1 extracellular domaincomprises the amino acid sequence of SEQ ID NO:5.
 13. The method ofclaim 11, wherein the soluble FGFR1 fusion protein comprises the aminoacid sequence of SEQ ID NO:8.
 14. The method of claim 11, wherein thesoluble FGFR1 fusion protein is administered at a dose of about 2 mg/kgbody weight to about 20 mg/kg body weight.
 15. The method of claim 14,wherein the soluble FGFR1 fusion protein is administered at a dose ofabout 8 mg/kg body weight to about 16 mg/kg body weight.
 16. The methodof claim 15, wherein the soluble FGFR1 fusion protein is administered ata dose of about 8 mg/kg body weight.
 17. The method of claim 15, whereinthe soluble FGFR1 fusion protein is administered at a dose of about 16mg/kg body weight.
 18. The method of claim 11, wherein the cancer isprostate cancer, breast cancer, colorectal cancer, lung cancer,endometrial cancer, head and neck cancer, laryngeal cancer, livercancer, renal cancer glioblastoma or pancreatic cancer.
 19. The methodof claim 11, wherein the human has an FGF-2 plasma concentration of atleast 10 pg/ml prior to the administration of the soluble FGFR1 fusionprotein.
 20. The method of claim 11, wherein the soluble FGFR1 fusionprotein is administered in combination with a chemotherapeutic agent ora VEGF antagonist.